1. Field of the Invention
This invention relates generally to the effective diagnosis and treatment of cancer patients and, more specifically, to radiopharmaceuticals for use in connection with radioimmunotherapy and radioimmunodection.
2. Background
Effective treatment of cancer patients by using radioimmunotherapy (RAIT) and diagnostics of malignant tumors by radioimmunodetection (RAID) requires drastic improvement of tumor-to-background (T/B) distribution of a radiolabel. T/B ratio is the principal parameter that determines tumoricidal action, sensitivity of tumor detection and, most importantly, systemic toxicity of the compound. Radioactive isotopes of metals are used very often in various modalities of RAIT and RAID in the form of conjugates with monoclonal antibodies (mAbs).
Attachment of the radioisotope to the targeting molecule is the most important part of the preparation of the radioconjugate. In some cases, a direct approach to radiolabeling of antibodies with therapeutic radionuclides can be adopted by reducing internal disulfide bonds within the antibody and allowing, for example, technetium or rhenium ions to react directly with the resultant sulfhydryl groups. However, labeling strategies usually rely on the utilization of a multidentate ligand capable of selective chelation of the desired radionuclide. In order to avoid the release of the metal ion in the circulation and inevitable radiation injuries to healthy organs, metal ions must be strongly complexed by a chelating agent.
Ideally, the chelated nuclide would be rapidly excreted via the urinary tract without intracellular retention, with intact conjugate accumulating and being retained selectively at the tumor site. This is however clearly not always the case, and in several instances, persistent and unwanted retention of metallic nuclides has been reported. As the in vivo behavior of a given nuclide-chelator complex cannot be predicted easily, the choice of potential multidentate ligands is guided by conventional chelation chemistry. Macrocyclic multidentate ligands which provide the highest thermodynamic stability of the complex are frequently used for preparation of immunoconjugates. The typical examples of chelating agents are diethylenetriaminepentaacetic anhydride (DTPA), (diethylenetriaminetetraacetic acid (DTTA), (1,4,7,10-tetraazacyclododecane-N,Nxe2x95x90,N,Nxe2x95x90-tetraacetic acid) (DOTA), ethylenediaminetetra(methylene phosphonate) (EDTMP), 1,1-hydroxyethylidine diphosphonate(HEDP) and derivatives thereof. For preparation of conjugated structures they are frequently functionalized in one of the side chains by xe2x80x94C6H4xe2x80x94NCS or other groups to form a covalent bond with proteins.
Entrapment of metal ion into a framework of muldidentate poly (amino carboxylates) such as DOTA, DTPA covalently attached to mAbs has been proven to provide thermodynamically stable compounds suitable for clinical trials. Nevertheless, results obtained for human patients and for animal models indicate that the applicability of these compounds is limited by the high level of radioactivity in blood, bone marrow, kidney and liver. This necessitates reduction of in-vivo decomplexation of metal ions and strengthening radiolabel-mAb bond.
The tumor uptake and overall biodistribution of the radiolabeled compound depends on the metabolism of the mAb and the strength of radionuclide-antibody link. Although there is a substantial room for improving targeting properties of mAbs, all studies indicate that the thermodynamic and kinetic stability of the chelate in vivo of is of primary importance for the design of clinically successful radiochemicals.
The equilibrium constant of chelation for the best chelates used in RAIT and RAID is comparable to that for some proteins present in blood. Therefore, chelates are inherently prone to slow exchange of radioactivity with tumor-indifferent proteins. When radiopharmaceuticals are administered intravenously, they encounter endogenous metal-binding proteins such as albumin in concentrations 100-1000-fold greater than that of the radiopharmaceutical. Loss of radiometal to serum proteins leads to accumulation of radioactivity in normal tissues, especially liver, spleen, and kidney, reducing the radioactivity available for tumor uptake. Stability of the radioimmunoconjugate is a critically important factor in determining its usefulness for in vivo applications. This fact was experimentally established for the majority of radionuclides.
For complexes of 111In and bleomycin, partial dissociation of the ion from bleomycin caused severe bone marrow toxicity and increased the background xcex3-radiation significantly impairing its therapeutic and imaging capabilities. The tumor-to-background ratio varied from 1.0 to 2.9. Even for one of the most widely used and the strongest 111In chelate with octadentate ligand DTPA (Kdiss=28.5) a high liver background has been observed. Most researchers agree that chelate instability is the primary reason for various immune mechanisms which contribute to accumulation of the radioactivity in non target organs. Recent study clearly indicate the direct dependence between the thermodynamic stability of various chelates (NTA, EGTA, EDTA, DTPA) bound to the B72.3 antibody and the tissue distribution and accumulated activity in blood and kidney.
Retention of 111In in normal tissues has been the major limitation in RAID applications. Improved clearance of 111In from liver tissue correlated with the strength of a labile linkage between the antibody and the chelator and with the structure of the chelator to provide for higher stability under in vivo conditions.
The loss of metal ion in circulation greatly limits the utilization of potent radionuclides with convenient energy and half-life characteristics such as 212Pb. The short decay of 212Pb time (10.6 hours, xcex2, 0.57 and 0.33 MeV) permits rapid clearance from the system. When conjugated to specific mAb 103 A, 212Pb-DOTA chelate was very efficacious at eliminating the tumor even in mice with large tumor burdens. However, due to lower pH values found in cells in which catabolism occurs, 212Pb escapes the chelator and is taken up in bone and marrow. Bone marrow toxicity in this case was so high that accumulation of 212Pb in bones was dose limiting and lethal. Loss of the specificity of radiometal deposition was observed for 46Sc, 67Ga, and 90Y when the ion was not strongly chelated to its ligand.
Incorporation of a radiometal label into the protein structure by using nitrogen and sulfur atoms of aminoacids (NS systems) is very popular for technetium and rhenium. Comparative stability of these chelates is quite high. Importantly, metal species can be incorporated in an anionic form that makes recomplexation by native proteins less probable. Nevertheless, 188Re-labeled mAbs were found to be a marginal agent for controlling tumor growth. The failure of 188Re-IgG in some tumor models may be related to the combination of apparent instability of the labeled product and its short physical half-life. Interestingly the dissociation of 188Re from the protein occurred more quickly that for other isotopes such as 88Y. The release of rhenium is likely to be assisted by reoxydation of the chelate to ReO4xe2x88x92 by dissolved oxygen.
Importantly, for yttrium (90Y, 88Y), lead (212Pb) and samarium (153Sm) the strength of the chelates must be particularly high. In ionic form these metals are capable of replacing Ca2+ ions from bone, which halts excretion of the radionuclide and causes augmented radiation damage to healthy tissues.
The nature of medical applications imposes multiple requirements on the chemical characteristics of a potential chelating ligand. It has to be (a) strong (multidentate) complexing agent for the metal ion, (b) hydrophilic to afford solubility in water, (c) nontoxic, (d) capable of incorporating into a protein structure without causing its denaturation. For virtually every single radionuclide one has to design a special chelating system. For example macrocyclic bifunctional chelating agents, in particular, DOTA, derivatives incorporating yttrium-90 and indium-111 have shown excellent kinetic stability under physiological conditions. However, the slow formation of yttrium-DOTA complexes presents a technical problem that can lead to low radiolabeling yields unless conditions are carefully controlled.
The most successful chelating agents for preparation of radioconjugates are the products of a complex organic synthesis. Quite representative examples of a synthetic procedure can be found in Brechbiel, M. W.; Gansow, O. A.; Atcher, R. W.; Schlom, J.; Esteban, J.; Simpson, D. E.; Colcher, D., Synthesis of 1-(P-isothiocyanatobenzyl) Derivatives of DTPA and EDTA. Antibody Labeling and Tumor Imaging Studies, Inorg. Chem., 1986, 25, 2772-2781. They consist of more than 6 consecutive steps. Synthetic sophistication not only increases the cost of the drug but also compromises the quality of the pharmaceutical by restricting how the label can be attached to mAb. Direct binding used for 99mTc and 186Re without specilly designed chelating agents simplifies the procedure. However, it cannot be applied for other metals and may induce conformational changes in mAb or its fragments.
Entrapment of a metal ion into a framework of multidentate complex yields a thermodynamically stable structure, but quite often, is slow to form. Typically, incorporation of a radioactive ion takes  greater than 3 hours. The fast forming chelates like those of EDTA, undergo rapid transchelation to blood proteins. Slow kinetics of metal incorporation decreases the overall activity of short half-life radionuclides and necessitates removal of unbound radioactive species.
It is thus an object of the present invention to provide a new method of preparation of radioimmunoconjugates to obtain more stable agents for radiotherapy and diagnostics.
In accordance with the present invention metal chelates are replaced with small (2-5 nm in diameter) clusters of metal sulfide nanoparticles bound to mAbs or other biological molecules via a bifunctional organic stabilizer. The core materials of the nanoparticles, i.e. transition metal sulfides, are virtually insoluble in aqueous solutions due to partially covalent character of the crystal lattice. Negligible concentration of ionic species prevents transchelation in serum and release of radioisotopes in circulation.
Nanoparticles (NPs) combine properties of bulk solids and relatively large molecules. The core of NPs is made of inorganic material and retains some physical and chemical properties of its bulk predecessor, while the solubility and chemical reactivity is determined by a thin, virtually monomolecular layer, of organic molecules adsorbed to NP. This layer passivates the surface of the solid and protects the NP from further growth. A chemical compound forming this layer is often referred to as a stabilizer. The inorganic core may contain 100 to 10,000 atoms depending on its diameter (1-10 nm). The size can be accurately assessed by the position of UV absorption peak that was shown to shift to shorter wavelengths for smaller NPs due to size-quantization effect. Very small size, uniformity of particles, and critical role of the surface distinguishes NPs from both colloidal and molecular systems.
Although a large variety of materials can be prepared in this form, predominantly metal sulfides and oxides have been studied for preparation of NPs. Organic thiols, R-SH are known to be excellent stabilizers for these materials due to formation of a relatively strong covalent R-S-M link, where M stands for metal atom on the surface of nanoparticle. Numerous methods have been proposed for preparation of NPs in solid and liquid media, as well as at interfaces. See, Fendler, J. H. Membrane-mimetic Approach to Advanced Materials. Advances in Polymer Science. Vol. 113. Springer-Verlag: Berlin, 1994; C. Luagdilok and D. Meisel, Size Control and Surface Modification of Colloidal Semiconductor Particles, Is. J. Chem, 1993, 33, 53; P. V. Kamat, Interfacial Charge Transfer Processes in Colloidal Semiconductor Systems, Prog. Reaction Kinetics, 1994, 19, 277. By choosing an appropriate stabilizer and reaction conditions one can alter the diameter of the core with a precision of a few Angstroms.
Unique properties of NPs, i.e. their intermediate position between single molecule and a bulk compound, have made them excellent candidates for various applications. So far, scientists viewed them as perspective candidates for optical and electronic devices, while medical applications of inorganic nanoparticles are very limited.
The term nanoparticle appears in medical literature quite often. In most cases it is used to describe objects of from 30 to 300 nm in diameter represented by vesicles, polymers or colloids. These species are used for drug delivery and for diagnostic purposes. Confusion of terms used in different fields of chemistry should not obscure the fundamental difference that drug delivery agents are expected to remain in the blood stream for relatively long time while the radioimmunoconjugates must have a rapid blood clearance which is hardly achievable with large species 50-300 nm in diameter. Accordingly, it is contemplated that the term nanoparticles in the present invention includes particles of a smaller diameter.
The use of inorganic clusters in the nanometer region has been limited so far to conjugates of Fe3O4 magnetic clusters proposed for MRJ contrast agents. Ideologically, these are the assemblies that are quite similar to MPC: a mAb or other targeting compound attached to the magnetic core causes magnetic material to accumulate in tumor tissue, which increases the contrast of MRI images. Magnetic conjugates were proven to be quite efficient agents for MRI. From chemical point of view, synthetic approaches must be entirely different because of difficulty of covalent modification of oxide surfaces used in magnetic imaging. Fe2O3 and Fe3O4 nanoparticles and coating material (polyelectrolytes) are held together mostly by electrostatic forces. Metal sulfides offer a possibility to form stronger and more precisely organized assemblies via covalent bonds. Importantly, the requirements to chemical stability in radioimmunoconjugates is substantially stricter than in magnetic imaging because of the high toxicity of radioactive materials.
Here and later, the term xe2x80x9csolutionxe2x80x9d shall be used rather than xe2x80x9cdispersionxe2x80x9d to describe nanoparticles in liquid media in order to emphasize the similarity with true molecular solutions. For nanoclusters 1-10 nm in diameter it is not only a valid but also a more appropriate terminology in accord with their chemical properties quite different from the common colloidal dispersions.
In connection with the present invention, instead of a multidentate ligand with a radioactive ion in the middle a radioactive nanoparticle is covalently linked to a biological vector molecule such as mAb, its fragment, peptide, or others. NPs are synthesized separately from a suitable isotope and then conjugated to biological molecules by using procedures similar to those for chelates. For example, terminal groups of stabilizer molecules coating NPs can be chemically modified to p-isothiocyanotobenzyl derivatives, which can be coupled to NH2 groups of lysine residues of proteins. Alternatively, NP-protein conjugates can be prepared via NP-biotin dyads (NP-B) to provide a synthetic route to preparation of heterogeneous conjugates combining two different bioactive moity. The new radiopharmaceuticals substantially improve the clinical properties of currently used radiolabeled compounds for the following reasons:
(1) The dissociation constants, K, for chelates are many orders higher than that of metal sulfides of clinical interest: logK for the strongest chelates is known to be  greater than xe2x88x9230, while logK(In2S3)=xe2x88x9273.24, logK(CuS)=xe2x88x9235.2, logK(Bi2S3)=xe2x88x9297, logK(Ag2S)=xe2x88x9249.7. When a radioactive metal is present in the body in the form of virtually insoluble largely covalent sulfide cluster, the formation of metal ions leading to transchelation by natural proteins is practically eliminated. Thus, the biodistribution of a radioactive material will not be compromised by the loss of chelated ion while in circulation.
(2) Numerous stabilizer molecules coating nanoparticles permit for several identical covalent bridges to be formed in one conjugation step making the overall stability of the conjugate substantially higher.
(3) Replacement of a metal complex with nanoparticles, which can be prepared for 10-20 min, eliminates long and expensive multistep procedure of chelate synthesis. Since inorganic core is much more tolerant to experimental conditions than elaborate organic molecules, surface activation and modification, should it be necessary, can be carried out as a one-pot synthesis without involvement of protective groups.
(4) Availability of multiple attachment points on the surface of nanoparticles enables design and preparation of radioimmunoconjugates with multiple targeting molecules. This will increase the uptake of the labeled compound by the cancerous cells and will promote a more uniform distribution of the drug within the tumor tissue. Conjugates of this type are currently unavailable by chelate labeling technique owing to complexity of the procedure and relatively short life-time of radionuclides.
Accordingly, application of nanoparticle/protein conjugates should significantly improve the detection limit of cancer, increase effectiveness of radiotherapy and reduce the overall radiation toxicity. Practical benefits of introducing nanoparticles in nuclear medicine also include the possibility of using low activity isotope sources. Since each nanoparticle contains 100-10,000 metal atoms, high radioisotope enrichment of the original stock solution is not required.
A better understanding of the present invention, its several aspects, and its objects and advantages will become apparent to those skilled in the art from the following detailed description, taken in conjunction with the attached drawings, wherein there is shown and described the preferred embodiment of the invention, simply by way of illustration of the best mode contemplated for carrying out the invention.